Method and a kit for the diagnosis of iga nephropathy

ABSTRACT

A method for the diagnosis of IgA nephropathy using a specific binding reaction comprising the steps: 
     a) preparing a substrate capable of binding fibronectin or IgA 
     b) contacting the substrate resulting from step a) with a sample of body fluid drawn from a patient subject to diagnosis to bind any fibronectin-IgA-complex present in said sample to the substrate, and 
     c) determining the presence of complex bound to the substrate using the reaction between the exposed part of such bound complex and a corresponding antibody thereto; and 
     a diagnostic kit for use in such diagnosis.

The present invention relates to a method for the diagnosis of IgAnephropathy using an antigen-antibody interaction, and the inventionalso includes a diagnostic kit for use in such a diagnosis.

Patients with primary IgA nephropathy, also known as Berger's disease,have been shown to have circulating IgA antibodies, binding to collagenIV prepared from glomerular basement membrane (GBM) (1). It was alsoshown that the IgA antibodies bound equally well to collagen I, II andIV and that denatured collagens bound antibodies most efficiently (1).In view of the wide distribution of the various collagens, it is ofinterest to note the coexistence with IgA nephropathy of symptoms fromextrarenal organs (2). Thus symptoms from skin, eye and joints are ofparticular interest since these structures contains collagen as a majorcomponent. Indeed one study reports presence of vascular IgA deposits inskin from patients with IgA nephropathy (3).

The association of exacerbations of clinical disease with upperrespiratory tract or gastrointestinal infections and the finding of IgAdeposits in the glomerular mesangium is well known. The finding ofincreased levels of IgA-bearing peripheral lymphocytes (4) as well asdecreased IgA-specific suppressor T cell activity (5) and increasedIgA-specific helper T-alfa cells (6) suggests an immunologicalmechanism.

Fibronectin, also known as cold insoluble globulin (CIq), that isaltered in many disease processes, is present both as a plasmaproteinand as a cell surface protein. The two forms differ slightly incomposition but share important functions as binding to gelatin(collagen), heparin, fibrin and cell surface receptors (7). Fibronectinis the major plasma component binding to gelatin (8) and this propertyas well as its heparin binding properties have been utilized in itsisolation (9). Interestingly fibronectin is present also in basementmembranes. It can be visualised by immunofluorescense not only in theglomerular basement membrane but also in the mesangium of healthyindividuals (10). Furthermore a concomitant increase of mesangialfibronectin and mesagial matrix was observed in patients with IgAnephropathy and Henoch-Schonleins purpura (10).

The present invention is based on the surprising discovery, that theIgA-antibodies are present in circulating immune complexes in patientswith primary IgA nephropathy together with fibronectin, and thisunexpected finding does, of course, explain the fact that theIgA-containing complexes have the ability of binding to collagen. Thisnew finding that the circulating immune complex contains, in addition toIgA, also fibronectin, enables the provision of both a method for thediagnosis of IgA nephropathy and a diagnostic kit for use in suchdiagnosis.

Accordingly, one object of the present invention is to provide a methodfor the diagnosis of IgA nephropathy using antigen-antibody interaction,and another object of the invention is to provide a diagnostic kit foruse in such diagnosis of IgA nephropathy.

The method of the present invention is characterized by the followingsteps:

a) preparing a substrate capable of binding fibronectin or IgA

b) contacting the substrate resulting from step a) with a sample of bodyfluid drawn from a patient subject to diagnosis to bind anyfibronectin-IgA-complex present in said sample to the substrate, and

c) determining the presence of complex bound to the substrate using thereaction between the exposed part of such bound complex and acorresponding antibody thereto.

In preparing such substrate capable of binding fibronectin preferredbinding agents are collagen, heparin, fibrin or anti-fibro nectin.According to this aspect of the invention the substrate is prepared insuch a way as to bind the fibronectin-IgA-complex through itsfibronectin component. This in turn means that the IgA-component of thecomplex is available for determining the presence of complex bound tothe substrate.

On the other hand the binding of the fibronectin-IgA-complex to thesubstrate can be directed to the IgA-part of the complex. In such casethere can be used as a binding agent either anti-IgA or a lectin, bothof which are capable of binding IgA. In this case the fibronectin partof the complex will be available for determining the presence of complexbound to the substrate.

The determination under step c) of the method of this invention can beof a quantitative nature and can be based on a conventional detectionsystem. Among such detection systems there may be mentioned those basedon enzymatic activity, or systems based on radiation emitted by aradioactive isotope or based on fluorescence.

The substrate used in binding the fibronectin-IgA-complex present in asample subject to diagnosis can be constituted by different objects,such as microtiter plates, laboratory test tubes, nitro-cellulose paper,plastic spheres or any other object suitable for the purpose.

Samples of body fluid drawn from a patient subject to the diagnosticmethod of the present invention may be constituted by for example humanblood, serum, plasma or saliva.

The invention also provides for a diagnostic kit for use in thediagnosis of IgA nephropathy, and such kit comprises:

a) an object capable of a binding fibronectin or IgA, and

b) reagent comprising a component which is capable of binding IgA orfibronectin and a detection component enabling determination offibronectin-IgA complex bound to the said object.

In the embodiments wherein the IgA part constitutes exposed part of thecomplex the object carries attached to the surface thereof heparin,collagen, fibrin or anti-fibronectin. In such case the reagentpreferably comprises lectin or antibody directed against IgA.

In the opposite case, wherein fibronectin is the exposed part of thecomplex the object carries attached to the surface thereof anti-IgA orlectin, the reagent comprising heparin, collagen, fibrin oranit-fibronectin.

In such diagnostic kit according to the invention the function of thedetection component may be based on enzymatic activity, radiationemitted by a radioactive isotope or fluorescence.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1a shows the binding of IgA to a heparin-Sepharose column;

FIG. 1b shows the binding of fibronectin (FN) to a heparin-Sepharosecolumn;

FIG. 1c shows the binding of the IgA-FN complex to a heparin-Sepharosecolumn;

FIG. 2 shows a diagram on the elution of proteins from an ordinary ionexchanger using a salt gradient;

FIG. 3a shows the binding of IgA to a jacalin-Sepharose column;

FIG. 3b shows the binding of fibronectin (FN) to a jacalin-Sepharosecolumn;

FIG. 3c shows the binding of IgA-FN complex to a jacalin-Sepharosecolumn;

FIG. 4a shows the separation of proteins using SDS-PAGE; and

FIG. 4b shows the separation of proteins using immunoblotting.

EXAMPLE 1 Antisera

Patient sera were from patients with primary IgA nephropathy(glomerulonephritis and dominating mesangial IgA deposits detected byimmunofluorescense microscopy of renal biopsy specimens). None of thepatients had signs of systemic lupus erythematosus (SLE),Henoch-Schonleins purpura or cirrhotic liver disease. Sera from healthyblood donors were used as controls.

EXAMPLE 2 Isolation of immune complexes

Antisera (1 ml) were dialysed against 0.05M Tris, 0.05% sodium azide pH7.4 before applied to a 10 ml Heparin-Sepharose CL-6B column (Pharmacia,Uppsala, Sweden). Material eluted from the column with 0.5M NaCl in thesame buffer was dialysed against 0.1M sodium phosphate, 0.05% sodiumazide pH 7.5 before chromatographed on a 1 ml Mono Q column (Pharmacia).Chromatography was performed using a LKB (Bromma, Sweden) HPLC systemwith low pressure mixing. Absorbance was measured at 280 nm with a LKB2151 variable wavelength detector. Fractions binding to collagen I andreacting with anti human IgA (Dakopatts, Hagersten, Sweden) were pooledand applied to a jacaline a (Pierce chemicals, Rockford, Ill.). IgAcontaining complexes were eluted with 0.2M melibiose in a buffercontaining 0.05M Tris, 0.15M NaCl, 0.05% sodium azide pH 7.5.

EXAMPLE 3 Preparation of collagen I

Collagen I was prepared by pepsin extraction from bovine flexor tendonas described by Vogel et al (12).

EXAMPLE 4 Electrophoresis

SDS-PAGE was performed as described by Laemmli (13) using 3-16% lineargradient gels. Gels were stained with silver according to the method ofMorrisey (14) omitting glutaraldehyde. Reduction was performed by adding2-mercaptoethanol to 0.2% v/v to samples prior to electrophoresis andboiling for two minutes.

EXAMPLE 5 ELISA

Antigen was coated to polystyrene 96-well microtiter plates (NUNCimmunoplate I, NUNC, Roskilde, Denmark). Fractions from the columns werecoated over night under non denaturing conditions using 0.05M sodiumcarbonate buffer at pH 9.6 containing 0.05% sodium azide. This wasfollowed by incubation for 1 hour with the same buffer also containing2% bovine serum albumin (blocking buffer) to prevent non specificbinding. Collagen I was coated under denaturing conditions using 6Mguanidine-HCl, 0.05M Tris-HCl pH 7.4 over night. In this case incubationwith blocking buffer was not needed. Sera were diluted in 0.01Mphosphate pH 7.5, 0.15M NaCl, 0.05% Tween 20 and 0.25M guanidine-HCl andincubated for 1 hour in the coated microtiter plate. Iqa antibodies weredetected by incubation with affinity purified anti human IgA alkalinephosphatase conjugate (Dako) for one hour. Fibronectin was detectedusing rabbit anti human fibronectin antiserua (Dako) followed by antirabbit-IgG alkaline phosphatase conjugate (Dako). Enzyme activity wasdetermined using p-nitrophenyl phosphate as the substrate. Microtiterplates were rinsed between each step with 0.15 M NaCl containing 0.05%Tween 20. All samples were analysed as triplicates. Absorbance wassonitored at 405 nm using a Titertek Multiskan photoseter.

EXAMPLE 6 Immunoblotting

Proteins were separated using SDS-PAGE and electrophoreticallytransferred to nitrocellulose paper (Schleicher and Schull,-Dassel,W.Germany) at 0.5 A for 4 hours. Non specific binding was prevented byincubation with blocking buffer for 1 hour. IgA antibodies were detectedusing peroxidase conjugated anti human IgA antiserum (Dako) andfibronectin was detected using rabbit anti human fibronectin followed byperoxidase conjugated anti rabbit antiserum. Enzyme activity wasdetermined with H₂ O₂ /diaminobenzidine (0.5 mg/al) (Fluka) in 0.05Msodium phosphate pH 7.5 containing cobalt chloride and ammonium nickelsulfate (15).

EXAMPLE 7 Isolation of CNBr-fragments

Since we have previously found that IgA antibodies from patients withIgA nephropathy bind to collagen I,II and IV (1) an attempt was made toisolate the specific epitope of bovine collagen I responsible for thebinding. Pepsin extracted collagen I was fragmented by the use of CNBrand the fragments were separated by cation exchange chromatography on aMono S column followed by gel chromatography on a TSK 83000SW column.Binding of antibodies to collagen I could be completely inhibited byfragments identified as the CB7 fragment of the alfa₁ chain and theCB3,5 fragment of the alfa₂ chain.

These fragments are known to contain the fibronectin binding domain(16). Thus, it appears that fibronectin is involved in the binding ofIgA antibodies to collagen. Indeed, as we found for patient IgAantibodies, Engvall et al (8) have previously shown that fibronectinbinds to both collagens I and II and that binding is enhanced whendenatured collagens are used. In, further support we could demonstrateby ELISA that anti fibronectin antibodies coated to a microtiter platecaused binding of IgA antibodies as well as fibronectin when incubatedwith serum from patients with primary IgA nephropathy.

When using serum from healthy blood donors as control no IgA antibodieswere bound to the anti fibronectin antibody coat. We therefore decidedto purify the complexes, in principle using affinity columns forfibronectin and IgA, respectively, in sequence.

EXAMPLE 8 Adsorption of fibronectin to heparin-Sepharose

Heparin-Sepharose (9) was used as the initial step for the purificationof fibronectin-IgA complexes from human serum. As demonstrated in ELISA(FIG. I), using the fractions as coat, expectedly most of the IgAantibodies were not retained on the heparin column. The fibronectinbound to the heparin column, and was eluted using 0.5M NaCl (FIG. 1).

It is known that fibronectin binds to collagen by its collagen bindingdomain located in the N-terminal part of the molecule (7,17). Thecollagen binding properties of fibronectin were utilized in an ELISA todemonstrate the presence of fibronectin-IgA immune complexes in theelute from the heparin column. Denatured collagen I (17) was used ascoat. Fractions from the heparin-Sepharose column were incubated in thecollagen coated microtiter wells. Bound IgA antibodies were detectedusing a specific antibody conjugate (FIG. 1). Only the fraction elutedfrom the heparin column by the use of NaCl contained antibody reactivityin the assay, apparently representing fibronectin-IgA immune complexesin which the IgA antibodies are bound to fibronectin and fibronectinmediates binding to collagen by its collagen binding domain.

The major portion of the IgA antibodies did not bind to the heparinsepharose and no collagen binding IgA antibodies were found in thisunbound fraction.

Contrasting results were obtained with a serum sample from a healthyblood donor. No immune complexes were detected neither in the fractionbound to the heparin-Sepharose nor in the unbound fraction (FIG. 1).

EXAMPLE 9 Binding of immune complexes to anion exchange column

Since heparin binds not only fibronectin, but other plasma proteins aswell (9) an ion exchange column was selected for the next step in thepurification procedure. Material eluted from the heparin column wasapplied to a Mono Q, anion exchange, HPLC column. Immune complexes,detected by the ELISA with collagen I coat, were eluted between0.22-0.24M NaCl (FIG. 2). The presence of free IgA antibodies (separatedfrom the immune complexes) demonstrates that a small proportion of theIgA binds to the heparin column and/or IgA is liberated due todissociation of immune complexes during the purification procedure.

EXAMPLE 10 Adsorption of the IgA component in immune complexes tojacalin-Sepharose

Fractions containing immune complexes (FIG. 2) were pooled andchromatographed on a jacalin-Sepharose column. Jacalin is a lectin thatcan be used for binding of IgA antibodies (18). This provides apossibility to separate the complexes from free fibronectin since thisstructure is not present on the fibronectin molecule. As expectedfibronectin is found both in the material not bound to the jacalincolumn and in the bound fraction that was eluted with melibiose, whileIgA antibodies are present only in the bound material (FIG. 3). Thefibronectin component of the immune complexes was bound to collagen I inan ELISA and fibronectin and IgA antibodies were identified (FIG. 3).The presence of components with mobilities corresponding to fibronectinand IgA in the material bound to the jacalin column was furtherdemonstrated with SDS-PAGE both with and without reduction (FIG. 4).Further proof for the identity of the components was obtained byimmunoblotting using anti-IgA and anti-fibronektin respectively (FIG.4). The major components visualized by SDS-PAGE was thus shown torepresent IgA and fibronectin, respectively.

The above examples show that patients with IgA nephropathy havecirculating immune complexes containing fibronectin and IgA antibodies.This is in agreement with a proposed immune complex mediated nature ofprimary IgA neprhopathy (11).

REFERENCES

1. Cederholm, B., Wieslander, J. and Heinegård, D. (1986) Proc. Natl.Acad. Sci. USA 83, 6151-6155.

2. Mustonen, J., Pasternack, A., Helin, H. and Nikkila, M. (1985) Am. J.Nephrol. 5, 150-157.

3. Baart De La Faille-Kuyper, E., Kater, L., Kuijten, R., Kooiker, C.,Wagenaar, S., Van Der Zouwen, P. and Mees, E. (1976) Kidney Int. 9,424-429.

4. Nomoto, Y., Sakai, H. and Arimori, S. (1979) Am. J. Clin. Pathol. 71,158-160.

5. Sakai, H., Nomoto, Y. and Arimori, S. (1979) Clin. Exp. Immunol. 38,243-298.

6. Sakai, H., Endoh, M. and Tomino, Y. (1982) Clin. Exp. Immunol. 50,77-82.

7. Hakomori, S., Fukuda, M., Sekiguchi, K. and Carter, W. (1984) In:Extracellular Matrix Biocemistry. Elsevier Science Publishing Co, NewYork, USA, 229-275.

8. Engvall, E., Rouslahti, E. and Miller, E. (1978) J. Exp. Med. 147:2,1584-1595.

9. Yamada, K (1982) Immunochemistry of the Extracellular Matrix 1,111-123.

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11. Coppo, R., Basolo, B., Piccoli, G., Mazzucco, G., Bulzomi, M.,Roccatello, D., De Marchi, M., Carbonara, A. and Di Belgiojoso, B.(1984) Clin. Exp. Immunol. 57, 583-590.

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We claim:
 1. A method for the diagnosis of IgA nephropathy by detectingfibronectin-IgA complex using a specific binding reaction, comprisingthe steps:a) preparing a solid support capable of specifically bindingfibronectin or IgA, b) contacting said solid support with a sample ofbody fluid drawn from a patient to bind fibronectin-IgA complex presentin said sample to said solid support by binding one member of saidfibronectin-IgA complex to said solid support, and c) determining thepresence of said complex bound to said solid support by adding aspecific binding reagent which binds to the other member of saidfibronectin-IgA complex not bound to said solid support in step b).
 2. Amethod according to claim 1, comprising preparing a solid support tobind to the fibronectin portion of said fibronectin-IgA complex whereinsaid support is coated with a substance selected from the groupconsisting of collagen, fibrin, and heparin.
 3. A method according toclaim 1, comprising preparing a solid support to bind to the fibronectinportion of said fibronectin-IgA complex wherein said support is coatedwith an anti-fibronectin substance.
 4. A method according to claim 1,comprising preparing a solid support to bind to the IgA-part of the saidfibronectin-IgA complex wherein said support is coated with an anti-IgAor IgA-binding lectin substance.
 5. A method according to any precedingclaim, wherein said body fluid is human blood, serum, plasma or saliva.6. The method according to claim 1 wherein said complex bound to saidsolid support is measured quantitatively using a label which is aradioactive isotope, a fluorescent moiety or a reagent which is part ofan enzyme detection system.
 7. A method according to claim 2 or 3,comprising binding the IgA-part of said complex to an enzyme-labeledanti-IgA or IgA-binding lectin.
 8. A method according to claim 4,comprising binding the fibronectin part of said complex to anenzyme-labeled anti-fibronectin, heparin, fibrin, or collagen.
 9. Amethod according to claim 1, wherein said solid support is a microtiterplate.
 10. A diagnostic kit for use in the diagnosis of IgA nephropathyby detecting fibronectin-IgA complex, comprising:a) a solid supportwherein anti-IgA or an IgA-binding lectin is attached to the surface ofsaid support, and b) a specific binding reagent conjugated to a label,wherein said specific binding reagent is heparin, collagen, fibrin oranti-fibronectin and wherein said label is a radioactive isotope, afluorescent moiety or a reagent which is part of an enzyme detectionsystem.
 11. A diagnostic kit for use in the diagnosis of IgA nephropathyby detecting fibronectin-IgA complex, comprising:a) a solid supportwherein anti-IgA or an IgA-binding lectin is attached to the surface ofsaid support, b) a specific binding reagent, wherein said specificbinding reagent is heparin, collagen, fibrin or anti-fibronectin, and c)an antibody which specifically binds to said specific binding reagent,said antibody conjugated with a label wherein said label is aradioactive isotope, a fluorescent moiety or a reagent which is part ofan enzyme detection system.